Abstract Our proposal investigates mechanisms of ?2-adrenergic receptor (?2AR) dysfunction that underlies airway hyper-responsiveness in asthma accounting for paradoxical loss of asthma control observed with frequent use of ?-agonist. Asthma pathology is associated with elevation of nitric oxide (NO) and indeed, patients with higher fraction of exhaled NO (FENO) have greater hyper-reactivity and airflow obstruction with paradoxical loss to ?-agonist challenge and re-challenge (Bonini et al., AJRCCM, 2013). ?-agonist activation of ?AR causes airway smooth muscle relaxation via cAMP in parallel leading to ?AR phosphorylation, desensitization and internalization. ?AR desensitization is well understood but mechanistically, little is known about ?2AR resensitization to recover active ?2ARs on cell surface. We recently uncovered mechanisms regulating ?2AR reactivation pathway wherein, dephosphorylation/reactivation of the ?2AR by protein phosphatase 2A (PP2A) is blocked by phosphoinositide 3-kinase ? (PI3K?), which serves to inhibit ?2AR resensitization limiting ?AR response in the cell to ?-agonist [PI3K?-PP2A axis] (Vasudeven et al., Mol Cell, 2011). It is not known whether desensitization and/or resensitization mechanisms underlie the loss in ?- agonist response with elevated NO in asthma. Contrary to NO mediating ?2AR desensitization via S- nitrosylation of proteins, our preliminary data in primary murine airway smooth muscle cells (ASMCs) reveal that NO causes ?2AR dysfunction not via desensitization rather, via resensitization pathways. NO activates PI3K? by S-nitrosylation of PI3K? blocking PP2A activity thereby, inhibiting ?2AR resensitization. Further, PP2A activity is determined by its methylation and we find that nitrosylated PI3K? inhibits PP2A activity in part by de-methylation. Consistently, primary ASMCs from asthmatic patients have markedly more intracellular phosphorylated-?2ARs, less cAMP response to ?-agonist, greater PI3K? activity and lesser PP2A activity compared to non-asthmatics at plasma membrane and endosome. Based on these findings, we hypothesize that NO inhibits ?2AR resensitization by nitrosylation/activation of PI3K? which inhibits PP2A and limits ?2AR reactivation in asthmatic airways with high NO. The result is cumulative loss of functional cell surface ?2ARs, pre-disposing cells to greater sensitivity for broncho-constrictors, i.e. airway hyper-reactivity (AHR). In strong support of our hypotheses, PI3K? null (PI3K?-/-) mice have minimal AHR, and AHR returns in the PI3K?-/- upon PP2A inhibition. To address our hypothesis, we propose to comprehensively determine the effects of elevated NO on ?2AR resensitization (Aim1) and its role in airway hyper-responsiveness (Aim 2), and to identify mechanisms of NO-mediated non-canonical activation of PI3K? that inhibits PP2A and ?2AR resensitization in asthma (Aim 3).